U.S. patent number 10,583,154 [Application Number 15/487,323] was granted by the patent office on 2020-03-10 for multi-dose compositions containing an antimicrobial polyamide or octenidine preservative.
This patent grant is currently assigned to Boehringer Ingelheim Animal Health USA Inc., GENZYME CORPORATION. The grantee listed for this patent is BOEHRINGER INGELHEIM ANIMAL HEALTH USA INC., GENZYME CORPORATION. Invention is credited to Pradeep K. Dhal, Celine Loss-Dunod, Alexis Guy Andre Lucien Parisot, Guillaume Rigaut.
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United States Patent |
10,583,154 |
Rigaut , et al. |
March 10, 2020 |
Multi-dose compositions containing an antimicrobial polyamide or
octenidine preservative
Abstract
The present invention relates to non-mercurial preservatives,
including antimicrobial polyamide polymers and octenidine, and to
methods of use thereof to produce preservative-containing
multi-dose formulations. The preservative-containing multi-dose
formulations exhibit resistance to one or more contaminating
microorganisms, and have advantageous properties with respect to
long term stability of biological and small molecule active
ingredients.
Inventors: |
Rigaut; Guillaume (Lyons,
FR), Loss-Dunod; Celine (Charly, FR),
Parisot; Alexis Guy Andre Lucien (Lyons, FR), Dhal;
Pradeep K. (Bridgewater, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
BOEHRINGER INGELHEIM ANIMAL HEALTH USA INC.
GENZYME CORPORATION |
Duluth
Cambridge |
GA
MA |
US
US |
|
|
Assignee: |
Boehringer Ingelheim Animal Health
USA Inc. (Duluth, GA)
GENZYME CORPORATION (Cambridge, MA)
|
Family
ID: |
59215875 |
Appl.
No.: |
15/487,323 |
Filed: |
April 13, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20170296577 A1 |
Oct 19, 2017 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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62322258 |
Apr 14, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
31/04 (20180101); A61K 47/186 (20130101); A61K
47/34 (20130101); A61K 31/444 (20130101); A61K
39/04 (20130101); C12N 7/00 (20130101); A61K
31/787 (20130101); A61P 31/12 (20180101); A61K
39/12 (20130101); A61K 39/0241 (20130101); A61K
2039/55566 (20130101); A61K 2039/552 (20130101); C12N
2750/10034 (20130101); A61K 2039/54 (20130101) |
Current International
Class: |
A61K
31/787 (20060101); A61K 39/02 (20060101); A61K
39/04 (20060101); A61K 47/34 (20170101); A61K
47/18 (20170101); A61K 39/00 (20060101); C12N
7/00 (20060101); A61K 39/12 (20060101); A61K
31/444 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Archana, editor. Vaccinophobia and Vaccine Controversies of the
21.sup.st Century. ISBN 978-1-4614-7437-1--Springer New York
Heidelberg Dordrecht London. cited by applicant .
Bis et al. Antimicrobial preservatives induce aggregation of
interferon alpha-2a: The order in which preservatives induce
protein aggregation is independent of the protein. International
Journal of Pharmaceutics 472 (2014) 356-361. cited by applicant
.
Guo et al. Preservative Formulation and Effectiveness in Oral
Solutions and Suspensions. PDA Metro Meeting, Feb. 15, 2011. cited
by applicant .
Meyer et al. Antimicrobial Preservative Use in Parenteral Products:
Past and Present. Journal of Pharmaceutical Sciences, vol. 96, No.
12, Dec. 2007. cited by applicant .
"7bq5a" Inclusion of Antioxidants and antimicrobial preservatives
in medicinal products. pp. 177-182. Directive 81/852/EEC as amended
Jul. 1997. cited by applicant.
|
Primary Examiner: Fubara; Blessing M
Attorney, Agent or Firm: Jarecki-Black; Judy Ezcurra;
John
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional application
No. 62/322,258, filed on 14 Apr. 2016, and incorporated herein by
reference in its entirety.
Claims
What is claimed is:
1. A preserved multi-dose formulation, wherein: the formulation
comprises at least two effective doses of one or more active
pharmaceutical ingredient (API) and a preservative effective amount
of a non-mercurial preservative selected from the group consisting
of ##STR00037## and combinations thereof, wherein n is an integer
from 1 to 400;_and combinations thereof; admixed in an oil-in-water
emulsion wherein the API is at least one antigen wherein the
formulation contains no mercurial preservative.
2. The preserved multi-dose formulation of claim 1, wherein the
polyamide has the following structure: ##STR00038## and wherein the
weight average molecular weight (WAMW) is from about 1.0 kDa to
about 15.0 kDa, as measured by size exclusion chromatography.
3. The preserved multi-dose formulation of claim 2, wherein the
polyamide has a WAMW from about 2.0 kDa to about 10 kDa.
4. The preserved multi-dose formulation of claim 2, wherein the
polyamide has a WAMW from about 2.5 kDa to about 7.76 kDa, or has a
WAMW of about 7.76 kDa.
5. The preserved multi-dose formulation of claim 1, wherein the
formulation comprises the antimicrobial polyamide polymer at a
concentration of between about 0.1 mg/mL to about 0.25 mg/mL of
formulation.
6. The preserved multi-dose formulation of claim 5, wherein the
formulation comprises the antimicrobial polyamide polymer at a
concentration of about 0.25 mg/mL of formulation.
7. The preserved multi-dose formulation of claim 5, wherein the
formulation further comprises an adjuvant.
8. The preserved multi-dose formulation of claim 5, wherein the
antigenicity of the immunogenic formulation is stable for not less
than 1 year, 1.5 years, 2 years or 2.5 years; and wherein the
multi-dose formulation remains stable despite at least ten
usages.
9. The preserved multi-dose formulation of claim 5, wherein,
following inoculation of the formulation with one or more
micro-organisms, the concentration of said micro-organisms is
reduced over time, wherein the formulation presents at least 1.0
log reduction from the initial micro-organism count at 24 hours, at
least 3.0 log reduction at 7 days from the previous value measured
and not more than 0.5 log increase at 28 days from the previous
value measured.
10. The preserved multi-dose formulation of claim 5, wherein,
following inoculation with one or more micro-organisms, the
formulation presents at least 2.0 log reduction from the initial
calculated count at 6 hours after inoculation, at least 3.0 log
reduction at 24 hours from the previous value measured and no
recovery at 28 days.
11. The preserved multi-dose formulation of claim 5, wherein the
one or more micro-organisms is selected from P. aeruginosa, S.
aureus, E. coli, B. subtilis and combinations thereof.
12. The preserved multi-dose formulation of claim 5, wherein the
formulation further comprises one or more of a buffer, a
cryoprotectant, a salt, a divalent cation, a non-ionic detergent
and an inhibitor of free radical oxidation.
13. The preserved multi-dose formulation of claim 5, wherein the
formulation is contained within a vial.
14. A pre-filled vaccine delivery device comprising the preserved
multi-dose formulation of claim 5.
15. The pre-filled vaccine delivery device of claim 14, wherein the
device is a syringe and the preserved multi-dose formulation is
formulated for intramuscular or subcutaneous injection.
Description
INCORPORATION BY REFERENCE
This application makes reference to US 2014/0271526 and US
2014/0275469, the disclosures of which are incorporated by
reference herein in their entireties. All documents cited or
referenced herein are likewise incorporated herein by reference,
and may be employed in the practice of the invention.
Field of the Invention
The disclosure relates to thimerosal-free preservatives for
multi-dose formulation. The preservatives include antimicrobial
polyamides and octenidine.
Background of the Invention
For multi-dose formulations including vaccines, preservatives are
required to minimize contamination of the composition of subsequent
doses after the first dose is used. The preservative must enable
the vaccine formulation to pass efficacy tests or antimicrobial
challenge tests according to the United States Pharmacopeia (USP)
in the U.S., British Pharmacopeia (BP), and European Pharmacopeia
(EP) in Europe.
Thimerosal is a commonly-used preservative in vaccines. Thimerosal
is a mercurial compound that is potentially irritating, and may
increase the chance of allergic reactions. Thimerosal is also
undesirable for the environment.
Accordingly, it would be advantageous to find new and safer
preservatives for vaccines to replace thimerosal. In this
application, Applicants disclose inter alia the use of
antimicrobial polyamide compounds and octenidine as preservatives
for vaccines. The highly effective preservatives are non-irritating
and contain no mercurial compounds.
SUMMARY OF THE INVENTION
In one aspect, the invention provides safe and effective
thimerosal-free preservative for multi-dose formulations including
vaccines. In an embodiment, the preservative may comprise water
soluble, antimicrobial, amine functional polyamides, having the
general structure as set forth in formulae I-V (below). These
polymers may be produced using the methods described in US
2014/0275469 and U.S. Pat. No. 9,326,994 B2 (to Genzyme).
In an embodiment, the polyamide is a compound of Formula (I):
##STR00001## wherein: i) m is 0, 1, 2, or 3; ii) n is 0, 1, 2, or
3; iii) o is 0, 1, 2, or 3; iv) p is 0 or 1; v) r is 0 or 1; vi) q
is an integer from 1 to 400; vii) Q.sup.x is NH,
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl; viii) Q.sup.y
is NH--R.sup.w, NH--CH.sub.2--R.sub.w, (C.sub.1-C.sub.10)alkyl, or
(C.sub.6-C.sub.14)aryl, wherein R.sup.w is absent or a
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.6-C.sub.14)aryl, or (C.sub.2-C.sub.9)heteroaryl; ix) R.sup.x
and R.sup.y are each independently a pharmaceutically acceptable
end group.
In another embodiment, the polyamide has the structure of Formula
(II):
##STR00002## wherein: i) m is 0, 1, 2, or 3; ii) n is 0, 1, 2, or
3; iii) o is 0, 1, 2, or 3; iv) p is 0 or 1; v) r is 0 or 1; vi) q
is an integer from 1 to 400; vii) Q.sup.x is NH,
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl; viii) Q.sup.y
is NH--R.sup.w, NH--CH.sub.2--R.sub.w, (C.sub.1-C.sub.10)alkyl, or
(C.sub.6-C.sub.14)aryl, wherein R.sup.w is absent or a
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.6-C.sub.14)aryl, or (C.sub.2-C.sub.9)heteroaryl; ix) R.sup.x
and R.sup.y are each independently a pharmaceutically acceptable
end group; x) X.sup.- is each independently a halo or any
pharmaceutically acceptable anion; xi) Y.sup.1 and Y.sup.2 are each
independently H or (C.sub.1-C.sub.10)alkyl optionally substituted
by one or more substituents selected from the group consisting of
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl,
(C.sub.1-C.sub.10)alkylamine, --S--O--(C.sub.1-C.sub.10)alkyl,
--O(O)C--(C.sub.1-C.sub.10)alkyl, --(C.sub.1-C.sub.10)alkyl-COOH,
(C.sub.3-C.sub.10)cycloalkyl-COOH, --(O)CH.sub.3, --OH, amide, a
dihydroxy group, represented by Formula (D),
##STR00003## wherein d is an integer from 0 to 25, or a
polyethylene glycol group, represented by Formula (E)
##STR00004## wherein e is an integer from 1 to 25.
In another embodiment, the polyamide has the structure of Formula
(III):
##STR00005## wherein: i) m is 0, 1, 2, or 3; ii) n is 0, 1, 2, or
3; iii) o is 0, 1, 2, or 3; iv) p is 0 or 1; v) r is 0 or 1; vi) q
is an integer from 1 to 400; vii) Q.sup.x is NH,
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl; viii) Q.sup.y
is NH--R.sup.w, NH--CH.sub.2--R.sub.w, (C.sub.1-C.sub.10)alkyl, or
(C.sub.6-C.sub.14)aryl, wherein R.sup.w is absent or a
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.6-C.sub.14)aryl, or (C.sub.2-C.sub.9)heteroaryl; i) R.sup.x
and R.sup.y are each independently a pharmaceutically acceptable
end group; ix) X.sup.- is each independently a halo or any
pharmaceutically acceptable anion; x) Y.sup.1 is H or
(C.sub.1-C.sub.10)alkyl optionally substituted by one or more
substituents selected from the group consisting of
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl,
(C.sub.1-C.sub.10)alkylamine, --S--O--(C.sub.1-C.sub.10)alkyl,
--O(O)C--(C.sub.1-C.sub.10)alkyl, --(C.sub.1-C.sub.10)alkyl-COOH,
(C.sub.3-C.sub.10)cycloalkyl-COOH, --(O)CH.sub.3, --OH, amide, a
dihydroxy group, represented by Formula (D),
##STR00006## wherein d is an integer from 0 to 25, or a
polyethylene glycol group, represented by Formula (E),
##STR00007## wherein e is an integer from 1 to 400.
In another embodiment, the polyamide has the structure of Formula
(IV):
##STR00008## wherein: i) u is 0, 1, 2, or 3; ii) v is 0, 1, 2, or
3; iii) q is an integer from 1 to 400; iv) Q.sup.x is NH,
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl; v) Q.sup.y is
NH--R.sup.w, NH--CH.sub.2--R.sub.w, (C.sub.1-C.sub.10)alkyl, or
(C.sub.6-C.sub.14)aryl, wherein R.sup.w is absent or a
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.6-C.sub.14)aryl, or (C.sub.2-C.sub.9)heteroaryl; vi) R.sup.x
and R.sup.y are each independently a pharmaceutically acceptable
end group.
In yet another embodiment, the polyamide has the structure of
Formula (V):
##STR00009## wherein: i) u is 0, 1, 2, or 3; ii) v is 0, 1, 2, or
3; iii) q is an integer from 1 to 400; iv) Q.sup.x is NH,
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl; v) Q.sup.y is
NH--R.sup.w, NH--CH.sub.2--R.sub.w, (C.sub.1-C.sub.10)alkyl, or
(C.sub.6-C.sub.14)aryl, wherein R.sup.w is absent or a
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.6-C.sub.14)aryl, or (C.sub.2-C.sub.9)heteroaryl; vi) R.sup.x
and R.sup.y are each independently a pharmaceutically acceptable
end group; vii) X.sup.- is independently a halo or any
pharmaceutically acceptable anion, xi) Y.sup.1 and Y.sup.2 are each
independently H or (C.sub.1-C.sub.10)alkyl optionally substituted
by one or more substituents selected from the group consisting of
(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl,
(C.sub.1-C.sub.10)alkylamine, --S--O--(C.sub.1-C.sub.10)alkyl,
--O(O)C--(C.sub.1-C.sub.10)alkyl, --(C.sub.1-C.sub.10)alkyl-COOH,
(C.sub.3-C.sub.10)cycloalkyl-COOH, --(O)CH.sub.3, --OH, amide, a
dihydroxy group, represented by Formula (D),
##STR00010## wherein d is an integer from 0 to 25, or a
polyethylene glycol group, represented by Formula (E)
##STR00011## wherein e is an integer from 1 to 400.
The preservative may also include octenidine. Octenidine
dihydrochloride is a cationic surfactant (at neutral or
physiological pH) and bis-(dihydropyridinyl)-decane derivative, and
prior to this disclosure, its primary use was as an antiseptic for
topical use.
##STR00012##
In another aspect, the disclosure provides methods of preparing
preserved multi-dose formulations, including vaccines, using the
disclosed polyamide polymers and octenidine.
It is an object of the invention to not encompass within the
invention any previously known product, process of making the
product, or method of using the product such that the Applicants
reserve the right and hereby disclose a disclaimer of any
previously known product, process, or method. It is further noted
that the invention does not intend to encompass within the scope of
the invention any product, process, or making of the product or
method of using the product, which does not meet the written
description and enablement requirements of the USPTO (35 U.S.C.
.sctn. 112, first paragraph) or the EPO (Article 83 of the EPC),
such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
These and other embodiments are disclosed or are obvious from and
encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
This disclosure contains no figures or drawings.
DETAILED DESCRIPTION OF THE INVENTION
It is noted that in this disclosure and particularly in the claims,
terms such as "comprises", "comprised", "comprising" and the like
can have the meaning attributed to it in U.S. Patent law; e.g.,
they can mean "includes", "included", "including", and the like;
and that terms such as "consisting essentially of" and "consists
essentially of" have the meaning ascribed to them in U.S. Patent
law, e.g., they allow for elements not explicitly recited, but
exclude elements that are found in the prior art or that affect a
basic or novel characteristic of the invention.
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V. published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
The term "about," as used herein, means approximately, in the
region of, roughly, or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
10%. In one aspect, the term "about" means plus or minus 20% of the
numerical value of the number with which it is being used.
Therefore, about 50% means in the range of 45%-55%. Numerical
ranges recited herein by endpoints include all numbers and
fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5,
2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all
numbers and fractions thereof are presumed to be modified by the
term "about."
The term "effective amount" as used herein means an amount of a
composition according to the present invention effective in
producing the desired veterinary effect.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicate otherwise. The word "or" means any one member of a
particular list and also includes any combination of members of
that list.
The term "animal" is used herein to include all mammals, birds and
fish. The animal as used herein may be selected from the group
consisting of equine, canine, feline, bovine, swine, ovine,
caprine, camelids, avian, primate, humans, and fish. The term
"animal" also includes an individual animal in any stage of
development, including embryonic and fetal stages. As used herein,
the term "pig" or "piglet" means an animal of porcine origin, while
"sow" refers to a female of reproductive age and capability.
The terms "polypeptide" and "protein" are used interchangeably
herein to refer to a polymer of consecutive amino acid
residues.
The term "nucleic acid", "nucleotide", and "polynucleotide" are
used interchangeably and refer to RNA, DNA, cDNA, or cRNA and
derivatives thereof, such as those containing modified backbones.
It should be appreciated that the invention provides
polynucleotides comprising sequences complementary to those
described herein. The "polynucleotide" contemplated in the present
invention includes both the forward strand (5' to 3') and reverse
complementary strand (3' to 5'). Polynucleotides according to the
invention can be prepared in different ways (e.g. by chemical
synthesis, by gene cloning etc.) and can take various forms (e.g.
linear or branched, single or double stranded, or a hybrid thereof,
primers, probes etc.).
As used herein, the term "antigen" or "immunogen" means a substance
that induces a specific immune response in a host animal. The
antigen may comprise a whole organism, killed, attenuated or live;
a subunit or portion of an organism; a recombinant vector
containing an insert with immunogenic properties; a piece or
fragment of DNA capable of inducing an immune response upon
presentation to a host animal; a polypeptide, an antigen, an
epitope, a hapten, or any combination thereof. Alternately, the
immunogen or antigen may comprise a toxin or antitoxin.
The term "immunogenic protein or peptide" as used herein includes
polypeptides that are immunologically active in the sense that once
administered to the host, it is able to evoke an immune response of
the humoral and/or cellular type directed against the protein.
Preferably the protein fragment is such that it has substantially
the same immunological activity as the total protein. Thus, a
protein fragment according to the invention comprises or consists
essentially of or consists of at least one epitope or antigenic
determinant. An "immunogenic" protein or polypeptide, as used
herein, includes the full-length sequence of the protein, analogs
thereof, or immunogenic fragments thereof. By "immunogenic
fragment" is meant a fragment of a protein which includes one or
more epitopes and thus elicits the immunological response described
above. Such fragments can be identified using any number of epitope
mapping techniques, well known in the art. For example, linear
epitopes may be determined by e.g., concurrently synthesizing large
numbers of peptides on solid supports, the peptides corresponding
to portions of the protein molecule, and reacting the peptides with
antibodies while the peptides are still attached to the supports.
Similarly, conformational epitopes are readily identified by
determining spatial conformation of amino acids such as by, e.g.,
x-ray crystallography and 2-dimensional nuclear magnetic
resonance.
The term "immunogenic protein or peptide" further contemplates
deletions, additions and substitutions to the sequence, so long as
the polypeptide functions to produce an immunological response as
defined herein. The term "conservative variation" denotes the
replacement of an amino acid residue by another biologically
similar residue, or the replacement of a nucleotide in a nucleic
acid sequence such that the encoded amino acid residue does not
change or is another biologically similar residue. In this regard,
particularly preferred substitutions will generally be conservative
in nature, i.e., those substitutions that take place within a
family of amino acids. For example, amino acids are generally
divided into four families: (1) acidic--aspartate and glutamate;
(2) basic--lysine, arginine, histidine; (3) non-polar--alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. Examples of conservative variations include the substitution
of one hydrophobic residue such as isoleucine, valine, leucine or
methionine for another hydrophobic residue, or the substitution of
one polar residue for another polar residue, such as the
substitution of arginine for lysine, glutamic acid for aspartic
acid, or glutamine for asparagine, and the like; or a similar
conservative replacement of an amino acid with a structurally
related amino acid that will not have a major effect on the
biological activity. Proteins having substantially the same amino
acid sequence as the reference molecule but possessing minor amino
acid substitutions that do not substantially affect the
immunogenicity of the protein are, therefore, within the definition
of the reference polypeptide. All of the polypeptides produced by
these modifications are included herein. The term "conservative
variation" also includes the use of a substituted amino acid in
place of an unsubstituted parent amino acid provided that
antibodies raised to the substituted polypeptide also immunoreact
with the unsubstituted polypeptide.
An "immunological response" to a composition or vaccine is the
development in the host of a cellular and/or antibody-mediated
immune response to a composition or vaccine of interest. Usually,
an "immunological response" includes but is not limited to one or
more of the following effects: the production of antibodies, B
cells, helper T cells, and/or cytotoxic T cells, directed
specifically to an antigen or antigens included in the composition
or vaccine of interest. Preferably, the host will display either a
therapeutic or protective immunological response such that
resistance to new infection will be enhanced and/or the clinical
severity of the disease reduced. Such protection will be
demonstrated by either a reduction or lack of symptoms normally
displayed by an infected host, a quicker recovery time and/or
lowered pathogen loads in the infected host.
The terms "recombinant" and "genetically modified" are used
interchangeably and refer to any modification, alteration or
engineering of a polynucleotide or protein in its native form or
structure, or any modification, alteration or engineering of a
polynucleotide or protein in its native environment or surrounding.
The modification, alteration or engineering of a polynucleotide or
protein may include, but is not limited to, deletion of one or more
nucleotides or amino acids, deletion of an entire gene,
codon-optimization of a gene, conservative substitution of amino
acids, insertion of one or more heterologous polynucleotides.
The terms "polyvalent vaccine or composition", "combination or
combo vaccine or composition" and "multivalent vaccine or
composition" are used interchangeably to refer to a composition or
vaccine containing more than one composition or vaccines. The
polyvalent vaccine or composition may contain two, three, four or
more compositions or vaccines. The polyvalent vaccine or
composition may comprise recombinant viral vectors, active or
attenuated or killed wild-type viruses, subunits
(proteins/antigens), DNA plasmids, or a mixture thereof.
As used herein, the term "inactivated vaccine" means a vaccine
composition containing an infectious organism or pathogen that is
no longer capable of replication or growth. The pathogen may be
bacterial, viral, protozoal or fungal in origin. Inactivation may
be accomplished by a variety of methods including freeze-thawing,
high pressure, chemical treatment (for example, treatment with
thimerosal, formalin, (betapropiolactone), BEI (binary
ethylenimine)), or any other chemical agent, including octenidine,
sonication, radiation, heat or any other convention means
sufficient to prevent replication or growth of the organism while
maintaining its immunogenicity.
Preservatives are commonly used in multi-dose formulations,
including animal vaccines, to prevent bacterial or fungal growth in
field conditions and are employed to minimize contamination of the
formulations during repeated (non-sterile) punctures into the vial.
Preservatives must pass antimicrobial effectiveness testing (AET),
as described in the US pharmacopeia. As disclosed herein,
Applicants performed the antimicrobial tests according to United
States Pharmacopeia (USP), British Pharmacopeia (BP), and European
Pharmocopeia (EP). Four test organisms were used: Candida albicans,
Pseudomonas aeruginosa, Staphylococcus aureus, and A. brasiliensis.
And while thimerosal and formol continue to play an important role
in veterinary vaccines, European authorities encourage the
development of vaccines that are free from those compounds. To
overcome potential future threats linked to a potential ban,
Applicants have identified two compounds and tested and confirmed
the efficacy of those as WFI solutions at different concentrations
for antimicrobial preservative testing according to Pharmacopoeia.
As disclosed herein, both the antimicrobial polyamide polymers and
octenidine are safe and effective vaccine preservatives and
bacterial inactivation agents.
In one aspect of the invention, vaccine preservative compositions
are provided, which comprise water soluble, antimicrobial, amine
functional polyamides or octenidine. The antimicrobial polyamide
may be any polyamide represented by Formula I, II, III, IV, or V.
In a particular embodiment, the polyamide is selected from one of
the twenty-five polymers (A-Y) listed in Table 1. In addition to
the particularly effective antimicrobial polyamide species
presented here, a skilled person can identify additional active
members of the disclosed genus with the application of non-routine
experimentation.
As illustrated in the Examples below, polyamides B and C are
particularly effective against a wide range of typical
contaminating pathogens, including S. aureus, P. aeruginosa, C.
albicans, and A. brasiliensis. mastitis-causing pathogens, at USP
and veterinarily acceptable levels.
Importantly, the values for "MW" in Table 1 indicate the "weight
average molecular weight," determined by size exclusion
chromatography (SEC), which is aqueous version of GPC. As such, as
used herein, for example, "polymer B" is intended to encompass
compositions containing polymer B having a weight average MW of
about 5 to about 15 kDa, about 5 to about 12 kDa, about 5 to about
10 kDa, about 6 to about 8.5 kDa, about 7.5 to about 8 kDa, or
about 7.76 kDa. Moreover, "MW" is intended to mean "weight average
molecular weight," unless otherwise expressly stated.
As indicated in Table 1, polymers B, C, and D each have the same
repeating structure (defined herein as poly(4,4-trimethylene
dipiperidine bispropanoic acid-diaminopropane)), but a different
weight average MW. Moreover, the MIC data show that polymers B, C,
and D tend to be comparably effective against the panel of
pathogens. Thus, Applicant has shown a wide range of
poly(4,4-trimethylene dipiperidine bispropanoic
acid-diaminopropane) MW grades are active antimicrobial agents
(i.e. MW grades from at least about 2.5 g/mol to at least about
10.6 g/mL).
As used herein, the polymers contain the following repeating units:
A [4,4-trimethylene dipiperidine bispropanoic
acid-4,4'-dipiperidine]; B-D [4,4-trimethylene dipiperidine
bispropanoic acid-diaminopropane]; E [2,2'-bipyrrolidine
bispropanoic acid-penta diamine]; G [4,4-trimethylene dipiperidine
bispropanoic acid-diaminopropane]; H [4,4-trimethylene dipiperidine
bispropanoic acid-N(2-aminoethyl)-diaminoethane]; I
[4,4'-trimethylene dipiperidine bispropanoic
acid-N(3-aminopropyl)1,3-propane diamine]; J [4,4'-trimethylene
dipiperidine bispropanoic acid-3,3'-diamino-N-methyl-dipropylamine;
K [4,4'-dipiperidine bispropanoic acid-2,2'-diamino diethylamine];
L [4,4'-dipiperidine bispropanoic acid-2,2'-diamino N-methyl
diethylamine]; M [4,4'-dipiperidine bispropanoic
acid-3,3'-diamino-dipropylamine]; N [4,4'-dipiperidine bispropanoic
acid-3,3'-diamino-N-methyl-dipropylamine]; O [4,4'-trimethylene
dipiperidine-1,3-diamninopropane-N,N'-di-3-propionic acid]; P
[4,4'-trimethylene dipiperidine bispropanoic
acid-N,N'-dimethyl-1,3-diaminopropane]; R [4,4-trimethylene
dipiperidine bispropanoic acid-4,4'-dipiperidine]; S
[4,4-trimethylene dipiperidine bispropanoic acid-diaminopropane];
and T [4,4'-trimethylene dipiperidine bispropanoic acid-N-glycidol
diethylene triamine].
In another aspect, the invention broadly provides multi-dose
formulations comprising active pharmaceutical ingredients (API),
including small molecule drugs (e.g. including famotidine), and
including biologicals (e.g. peptides, including insulin). Like
multi-dose vaccine formulations, multi-dose formulations containing
API are subjected to contamination or inoculation with
microorganisms upon repeated needle punctures. As such, in some
embodiments, the invention provides preserved multi-dose
API-containing formulations. API routinely included in
preservative-containing, multi-dose formulations encompass, but are
not limited to, the following:
Biologic therapeutics: Antivenin (Micrurus fulvius) (Equine
Origin), Antivenin (Crotalidae) (Equine Origin), Antivenin
(Latrodectus mactans), Calcitonin-salmon injection, Desmopressin
acetate, Etanercept, Epoetin alfa (recombinant), Epoetin alfa
(recombinant), Follitropin alfa injection, Insulin aspart
(recombinant), 70% insulin aspart protamine suspension, 30% insulin
aspart injection (rDNA origin), Insulin glargine [rDNA origin]
injection, Insulin glulisine (rDNA origin) injection, Regular U-500
(Concentrated) (insulin human injection USP [rDNA] origin), Insulin
Lispro Injection (rDNA origin), 75% Insulin Lispro Protamine
Suspension and 25% Insulin Lispro Injection (rDNA origin),
Interferon alfa-n3 (human leukocyte derived), Interferon alfa-2b,
recombinant, Interferon alfa-2a, recombinant, Leuprolide acetate
injection, Octreotide acetate injection, peginterferon alfa-2a, Rho
(D) Immune Globulin (human), Rho (D) Immune Globulin (human),
Sargramostim (recombinant), Somatropin (rDNA origin), Trastuzumab
and Tuberculin purified protein derivative.
Vaccines: LEPTODOG.RTM., RECOMBITEK.RTM. (inactivated vaccine
against canine leptospirosis), rabies vaccines, including
ALURABIFFA.RTM. and RABISIN.RTM. (inactivated rabies vaccines
adjuvanted), SYNTOXAN.RTM. line (clostridia vaccine), HYORESP.RTM.
(inactivated vaccine for pigs against mycoplasma hyopneumoniae),
CIRCOVAC.RTM. (inactivated vaccine against porcine circovirus 2
(PCV2)), PARVORUVAX.RTM. (combo vaccine against porcine parvovirus,
reovirus and erysipelothrix), Avian killed vaccines (for chicken
& turkeys): BIGOPEST.RTM., BINEWVAX.RTM., COR2.RTM.,
GUMBOPEST.RTM., GUMBORIFFA.RTM., IMOPEST.RTM., TUR3.RTM.,
FLUVAC.RTM., GALLIMUNE.RTM. range for Breeder/Layer/Broiler:
vaccines against Newcastle Disease (ND), (IBD) Infectious
bronchitis, Flu H9N2, Flu H5N9, (EDS) egg drop syndrom, Swollen
head syndrome, REOvirus, (ART) avian rhinotracheitis, (IB)
infectious Bursal disease, Diphtheria and tetanus toxoids and
acellular pertussis adsorbed, Diphtheria and tetanus toxoids and
acellular pertussis adsorbed, hepatitis B (recombinant) and
inactivated poliovirus vaccine combined, Diphtheria and tetanus
toxoids and acellular pertussis vaccine adsorbed, Hepatitis A
vaccine, inactivated, Hepatitis A inactivated and hepatitis B
(recombinant) vaccine, Hepatitis B vaccine (recombinant) and
Pneumococcal vaccine polyvalent.
Small molecules: Apomorphine hydrochloride injection, Enalaprilat,
Enoxaparin sodium injection Intravenous (conjugated estrogens, USP)
for injection, Famotidine, Fulvestrant injection, Haloperidol,
Hydrocortisone sodium phosphate Hydrocortone Phosphate Injection,
Hydromorphone hydrochloride, Metaraminol bitartrate, Nalbuphine
hydrochloride, Methylprednisolone acetate injectable suspension,
USP, Ondansetron hydrochloride, Penicillin G benzanthine
suspension, Phytonadione, Sodium ferric gluconate complex in
sucrose injection, Testosterone Enanthate Injection USP, Multiple
Dose Vial, Medroxyprogesterone acetate injectable suspension, USP,
Medroxyprogesterone acetate and estradiol cypionate injectable
suspension, Flumazenil injection, Dolasetron mesylate injection and
Cimetidine hydrochloride injection, Streptomycin sulfate, USP.
The present invention thus provides novel and non-obvious
antimicrobial polyamide compositions, and methods of using same for
treating and preventing mastitis in non-human animals. The methods
generally comprise administering to an infected animal an effective
amount of the veterinary composition to eliminate or cure,
completely or substantially, mastitis-causing pathogen(s). As
detailed below, the polyamide compounds are also highly active
against a broad range of other, significant, human and animal
pathogens. Moreover, the polyamides have been shown to be
well-tolerated in mice and rats. For example, the maximum tolerated
dose for 4,4'-trimethylene dipiperidine bispropanoic
acid-co-1,3-diamino propane was about 5 mg/kg (IP) and 40 mg/kg
(IV).
TABLE-US-00001 TABLE 1 Twenty-five Antimicrobial Amine Functional
Polyamides. "MW" = weight average molecular weights ID MW #
Structure (kDa) A ##STR00013## 10.6 B ##STR00014## 7.76 C
##STR00015## 3.35 D ##STR00016## 2.5 E ##STR00017## 3.0 F
##STR00018## 4.2 G ##STR00019## 2.0 H ##STR00020## 3-10 I
##STR00021## 5.0 J ##STR00022## 5.0 K ##STR00023## 7.0 L
##STR00024## 5.0 M ##STR00025## 5.4 N ##STR00026## 5.5 O
##STR00027## 10.0 P ##STR00028## 5.4 Q ##STR00029## 7.5 R
##STR00030## 3-10 S ##STR00031## 4.9 T ##STR00032## 4.5 U ~10 V
##STR00033## 8.4 W ~10 X 5-10 Y 5-10
"Substituted" means the substitution of a carbon in alkyl,
heterocyclic or aryl groups with one or more non-carbon
substituent. Non-carbon substituents are selected from nitrogen,
oxygen and sulfur.
"Unsubstituted" means the group is comprised of only hydrogen and
carbon.
The term "polymer" means a molecule comprised of repeating units.
The term "repeat unit" or "monomer" means a group in a polymer that
repeats or appears multiple times in a polymer. A polymer may be a
copolymer if the repeating units or "comonomers" are chemically and
structurally different from one another.
The term "pharmaceutically acceptable anion" means an anion that is
suitable for pharmaceutical use. Pharmaceutically acceptable anions
include but are not limited to halides, carbonate, bicarbonate,
sulfate, bisulfate, hydroxide, nitrate, chloride, phosphate,
persulfate, sulfite, acetate, ascorbate, benzoate, citrate,
dihydrogen citrate, hydrogen citrate, oxalate, succinate, tartrate,
taurocholate, glycocholate, and cholate.
The term "pharmaceutically acceptable end group" means an end group
that is suitable for pharmaceutical use. Examples of
pharmaceutically acceptable end groups include but are not limited
to H, (C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.9)heteroalkyl,
(C.sub.3-C.sub.10)cycloalkyl, (C.sub.2-C.sub.9)heterocycloalkyl,
(C.sub.6-C.sub.14)aryl, (C.sub.2-C.sub.9)heteroaryl,
(C.sub.1-C.sub.10)alkylamine, --O(O)C--(C.sub.1-C.sub.10)alkyl,
(C.sub.1-C.sub.10)alkyl-COOH, (C.sub.3-C.sub.10)cycloalkyl-COOH,
--(O)CH.sub.3, --OH, amide, a guanidino group, a guanidinium
chloride group, a guanidinobenzene group, a dihydroxy group, and a
polyethylene glycol group.
The term "effective amount" of a disclosed amine functional
polyamides is a quantity sufficient to achieve a therapeutic and/or
prophylactic effect on the particular condition being treated, such
as an amount which results in the prevention or a decrease in the
symptoms associated with mastitis. The precise amount of the
disclosed amine functional polyamides that is administered will
depend on the type and severity of mastitis or infection being
treated and on the characteristics of the animal, such as general
health, age, body weight and tolerance to drugs.
In one embodiment of the method, an effective amount of the
polyamide or octenidine is added to one or more antigens to form a
stable immunological composition.
According to another aspect of the present invention, a use of an
antimicrobial amine functional polyamide for preserving a vaccine
formulation is provided.
Preserved Vaccine Compositions
In accordance with the present invention, the preserved vaccine
composition comprises a water soluble, antimicrobial polyamide
polymer. Particularly effective polyamides include polymers B, C,
D, U, and T.
As used herein, the term "preserving effective amount" refers to a
concentration of preservative sufficient to prevent the growth of
contaminating microorganisms in a multiuse vaccine formulation.
The preservative-containing immunogenic composition of the present
invention may be resistant to several pathogens including E. coli,
Klebsiella spp., Enterobacter spp., Salmonella spp., Citrobacter
spp., Serratia spp., Shigella spp., Edwardsiella spp., Hafnia spp.,
Morganella spp., Providencia spp., Yersinia spp., Staphylococcus
aureus, Staphylococcus spp., Pseudomonas spp., Streptococcus
agalactiae, Streptococcus dysgalactiae, Streptococcus spp.,
Enterococci, Corynebacterium spp., Arcanobacterium spp.,
Actinomyces spp., Mycobacterium spp., Prototheca spp., Mycoplasma
spp., Erwinia spp., Lactobacillus spp., among others.
In an embodiment, the preservative-containing composition comprises
a Borrelia burgdorferi bacterial extract (e.g. antigens disclosed
in any of U.S. Pat. Nos. 5,523,089, 5,582,990, 5,688,512, 5,777,095
and 6,083,722, each to Symbicom). In one embodiment, the antigen
comprises an expression vector containing OspA (e.g. as disclosed
in U.S. Pat. No. 6,159,477 or 6,368,603, both to Merial).
In another embodiment, the preservative-containing multi-dose
formulation comprises an E. Coli bacterin-toxoid (e.g. Merial's
J-VAC.RTM.). In other embodiments, multi-dose formulation comprises
one or more of the antigens in the following vaccine compositions:
LEPTODOG.RTM., RECOMBITEK.RTM. (inactivated vaccine against canine
leptospirosis), rabies vaccines, including ALURABIFFA.RTM. and
RABISIN.RTM. (inactivated rabies vaccines adjuvanted),
SYNTOXAN.RTM. line (clostridia vaccine), HYORESP.RTM. (inactivated
vaccine for pigs against mycoplasma hyopneumoniae), CIRCOVAC.RTM.
(inactivated vaccine against porcine circovirus 2 (PCV2)),
PARVORUVAX.RTM. (combo vaccine against porcine parvovirus, reovirus
and erysipelothrix), Avian killed vaccines (for chicken &
turkeys): BIGOPEST.RTM., BINEWVAX.RTM., COR2.RTM., GUMBOPEST.RTM.,
GUMBORIFFA.RTM., IMOPEST.RTM., TUR3.RTM., FLUVAC.RTM.,
GALLIMUNE.RTM. range for Breeder/Layer/Broiler: vaccines against
Newcastle Disease (ND), (IBD) Infectious bronchitis, Flu H9N2, Flu
H5N9, (EDS) egg drop syndrom, Swollen head syndrome, REOvirus,
(ART) avian rhinotracheitis, (IB) infectious Bursal disease,
In an embodiment, the composition is a multi-dose vaccine
composition. In some embodiments, the multi-dose vaccine
composition is a monovalent compositions. In other embodiments, the
multi-dose vaccine composition is a multi-valent vaccine
composition.
In embodiments where the multi-dose composition is a monovalent
composition, the antigen may include one antigen, including one
selected from an antigen present in one of the following vaccines:
LEPTODOG.RTM., RECOMBITEK.RTM. (inactivated vaccine against canine
leptospirosis), rabies vaccines, including ALURABIFFA.RTM. and
RABISIN.RTM. (inactivated rabies vaccines adjuvanted),
SYNTOXAN.RTM. line (clostridia vaccine), HYORESP.RTM. (inactivated
vaccine for pigs against mycoplasma hyopneumoniae), CIRCOVAC.RTM.
(inactivated vaccine against porcine circovirus 2 (PCV2)), and
PARVORUVAX.RTM. (combo vaccine against porcine parvovirus, reovirus
and erysipelothrix).
In embodiments where the multi-dose composition is a multivalent
composition, the composition contains at least two antigens,
including antigens selected from among the following:
canarypox-vectored canine distemper, adenovirus type 2,
parainfluenza, parvovirus (MLV), Leptospira Canicola bacterin and
Leptospira icterohaemorrhagiae Bacterin (e.g. Merial's
RECOMBITEK.RTM. C6). In another embodiment, the multi-valent
composition contains: Encephalomyelitis-Influenza Vaccine, Eastern
and Western, Killed Virus, Tetanus Toxoid (i.e. Merial's EQUINE
EWTF).
In view of the above, it will be seen that the several objects of
the invention are achieved and other advantageous results attained.
As various changes could be made in the above formulations,
products, and processes without departing from the scope of the
invention, it is intended that all matter contained in the above
description and shown in the accompanying tables shall be
interpreted as illustrative and not in a limiting sense.
When introducing elements of the present invention or the preferred
embodiments(s) thereof, the articles "a", "an", "the" and "said"
are intended to mean that there are one or more of the elements.
The terms "comprising", "including" and "having" are intended to be
inclusive and mean that there may be additional elements other than
the listed elements. Moreover, the term "consisting essentially of"
is intended to mean that there may be additional elements other
than the listed elements, but not ones that would be considered
"active ingredients" (e.g. non-active excipients). And finally, the
term "consisting of" is intended to mean that only the listed
elements are included.
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V. published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
In one aspect, the invention provides a method of preserving a
multi-dose formulation, including those containing therapeutic
biologics, vaccines, and small molecules, comprising mixing the
multi-dose formulations with a non-mercurial preservative, selected
from a polyamide polymer and octenidine, to form a preserved
multi-dose formulation, thereby preserving the multi-dose
formulation.
In some embodiments, the invention provides a method of preparing a
preserved multi-dose formulation, the method comprising, consisting
essentially of, or consisting of the following steps: a) providing
at least two effective doses of one or more active pharmaceutical
ingredient (API); b) providing a preservative effective amount of a
non-mercurial preservative selected from one or more antimicrobial
polyamide polymer(s), octenidine and combinations thereof; c)
combining the API and the non-mercurial preservative with an
aqueous and/or oily component to form a preserved multi-dose liquid
formulation; d) optionally providing one or more excipient; e)
optionally providing a container for containing the liquid
formulation; f) optionally sterilizing and transferring the liquid
formulation to the container; and g) optionally freeze-drying,
lyophilizing and/or vitrifying the liquid formulation; thereby
producing the preserved multi-dose formulation; and wherein the
formulation contains no mercurial preservative.
The foregoing steps may be practiced in any reasonable order. For
example, the non-mercurial preservative(s), API(s) and optional
excipient may be placed into a mixing vessel containing an aqueous
and/or oily component. Once thoroughly mixed into a solution,
suspension, suspoemulsion, emulsion or micellar solution, the
preserved liquid formulation may then be filter-sterilized and
aseptically transferred into sterile containers, including vaccine
vials. The excipient may include any pharmaceutically or
veterinarily acceptable excipient, including those that improve the
solubility of the non-mercurial preservative(s). Moreover, any
suitable preserved liquid formulation may be produced.
In particular embodiments, the liquid formulation may be an aqueous
solution, a micellar solution, a swollen micellar solution, an
oil-in-water emulsion, a water-in-oil emulsion or a suspoemulsion.
In general, for preserved immunological or other biologic
formulations, the antigen or other biologic is dissolved within the
aqueous phase of a multi-phasic liquid formulation. The emulsions
may be microemulsions or "submicron" emulsions. Submicron emulsions
are generally referred to herein as "nanoemulsions."
Moreover, for the preservative to be deemed to be present in the
preserved multi-dose formulations in a "preservative effective
amount," the formulations should be protected from inoculation with
micro-organisms as disclosed herein. For example, if the amount of
bacteria inoculated into a preserved multi-dose formulation
decreases over time, the non-mercurial preservative is deemed to be
present in a preservative effective amount. In another example, the
preservative is present in a preservative effective amount when the
preserved formulation passes the tests detailed in Examples 1, 2
and/or 3.
In another aspect, the invention provides a preserved multi-dose
formulation, wherein: a) the formulation comprises at least two
effective doses of one or more active pharmaceutical ingredient
(API) and a preservative effective amount of a non-mercurial
preservative selected from one or more antimicrobial polyamide
polymer(s), octenidine and combinations thereof; or b) the
formulation is produced according to the disclosed method of
preparing a preserved multi-dose formulation; and wherein the
formulation contains no mercurial preservative.
In some embodiments, the preserved multi-dose formulation comprises
a polyamide polymer selected from:
##STR00034## and combinations thereof.
In some embodiments, the preserved multi-dose formulation comprises
a polyamide polymer having the following structure:
##STR00035## and wherein the weight average molecular weight (WAMW)
is from about 1.0 kDa to about 15.0 kDa, as measured by size
exclusion chromatography. In other embodiments, the polyamide may
have a WAMW from about 2.0 kDa to about 10 kDa. In still other
embodiments, the polyamide may have a WAMW from about 2.5 kDa to
about 7.76 kDa, or has a WAMW of about 7.76 kDa.
In another aspect, the invention provides an immunogenic
composition comprising at least one antigen, and further comprising
at least about 0.01 mg/mL of polyamide polymer or at least about
0.1 mg/mL octenidine. In the immunogenic composition, the polyamide
polymer or octenidine may be present at a concentration of between
about 0.05 mg/mL to about 5 mg/mL or between about 0.1 mg/mL to
about 0.25 mg/mL. In some embodiments, the polyamide polymer or
octenidine is present at a concentration of about 0.1 mg/mL, about
0.125 mg/mL or about 0.25 mg/mL. Now that the invention has been
disclosed, the skilled person may make routine adjustments to the
concentrations of these safe and effective non-mercurial
preservatives to achieve the desired preservative efficacy.
In still other embodiments, the immunogenic composition comprises
not less than about 0.1 mg/mL of the polyamide polymer. In some
embodiments, the composition comprises not less than about 0.1
mg/mL of the octenidine.
In some embodiments, the immunogenic composition further comprises
an adjuvant. Suitable adjuvants for use in the practice of the
present invention are (1) polymers of acrylic or methacrylic acid,
maleic anhydride and alkenyl derivative polymers, (2)
immunostimulating sequences (ISS), such as oligodeoxyribonucleotide
sequences having one or more non-methylated CpG units (Klinman et
al., 1996; WO98/16247), (3) an oil in water emulsion, such as the
SPT emulsion described on p 147 of "Vaccine Design, The Subunit and
Adjuvant Approach" published by M. Powell, M. Newman, Plenum Press
1995, and the emulsion MF59 described on page 183 of the same work,
(4) cation lipids containing a quaternary ammonium salt, e.g., DDA
(5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7)
saponin (including from the bark of Quillaja saponaria), (8) other
adjuvants discussed in any document cited and incorporated by
reference into the instant application, or (9) any combinations or
mixtures thereof.
The oil-in-water emulsion (3), which is especially appropriate for
viral vectors, can be based on: light liquid paraffin oil (European
pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil
resulting from the oligomerization of alkenes, e.g. isobutene or
decene, esters of acids or alcohols having a straight-chain alkyl
group, such as vegetable oils, ethyl oleate, propylene glycol,
di(caprylate/caprate), glycerol tri(caprylate/caprate) and
propylene glycol dioleate, or esters of branched, fatty alcohols or
acids, especially isostearic acid esters. The oil is used in
combination with emulsifiers to form an emulsion. The emulsifiers
may be nonionic surfactants, such as: esters of on the one hand
sorbitan, mannide (e.g. anhydromannitol oleate), glycerol,
polyglycerol or propylene glycol and on the other hand oleic,
isostearic, ricinoleic or hydroxystearic acids, said esters being
optionally ethoxylated, or polyoxypropylene-polyoxyethylene
copolymer blocks, such as Pluronic, e.g., L121.
Among the type (1) adjuvant polymers, preference is given to
polymers of cross linked acrylic or methacrylic acid, especially
cross linked by polyalkenyl ethers of sugars or polyalcohols. These
compounds are known under the name carbomer (Pharmeuropa, vol. 8,
no. 2, Jun. 1996). One skilled in the art can also refer to U.S.
Pat. No. 2,909,462, which provides such acrylic polymers cross
linked by a polyhydroxyl compound having at least three hydroxyl
groups, preferably no more than eight such groups, the hydrogen
atoms of at least three hydroxyl groups being replaced by
unsaturated, aliphatic radicals having at least two carbon atoms.
The preferred radicals are those containing 2 to 4 carbon atoms,
e.g. vinyls, allyls and other ethylenically unsaturated groups. The
unsaturated radicals can also contain other substituents, such as
methyl. Products sold under the name Carbopol (BF Goodrich, Ohio,
USA) are especially suitable. They are cross linked by allyl
saccharose or by allyl pentaerythritol. Among them, reference is
made to Carbopol 974P, 934P and 971P.
In some embodiments, the antigenicity of the immunogenic
composition is stable for not less than 1 year, 1.5 years, 2 years
or 2.5 years.
In other embodiments, following inoculation of the immunogenic
composition (or other preserved formulation) with one or more
micro-organisms, the concentration of said micro-organisms is
reduced over time owing to the presence of the preservative. When
this reduction is observed, the non-mercurial preservative is
deemed to be present in a "preservative effective amount."
In some embodiments, following inoculation with one or more
bacteria strains, the immunogenic composition (or other preserved
formulation) presents at least 1.0 log reduction from the initial
micro-organism count at 24 hours, at least 3.0 log reduction at 7
days from the previous value measured and not more than 0.5 log
increase at 28 days from the previous value measured.
In still other embodiments, following inoculation with one or more
bacteria strains, the immunogenic composition (or other preserved
formulation) presents at least 2.0 log reduction from the initial
calculated count at 6 hours after inoculation, at least 3.0 log
reduction at 24 hours from the previous value measured and no
recovery at 28 days.
In some embodiments, the one or more micro-organisms are selected
from P. aeruginosa, S. aureus, E. coli and B. subtilis.
In other embodiments, the immunogenic composition (or other
preserved formulation) is inoculated multiple times. For example, a
second inoculation may occur at 6 hours following an initial
inoculation, a third inoculation may occur at 24 hours following
the initial inoculation, a fourth inoculation may occur at 7 days
following the initial inoculation and a fifth inoculation may occur
at 14 days following the initial inoculation.
In some embodiments, the immunogenic composition (or other
preserved formulation) may further comprise one or more of a
buffer, a cryoprotectant, a salt, a divalent cation, a non-ionic
detergent, and an inhibitor of free radical oxidation.
In another aspect, the invention provides a vial containing an
immunogenic composition (or other preserved formulation) as
disclosed herein.
In some embodiments, the vial may contain more than two doses of
the immunogenic composition (or other preserved formulation).
In another aspect, the disclosure provides a preserved multi-dose
formulation made according to the disclosed methods.
In another aspect, the disclosure provides an immunogenic
composition comprising at least one antigen, and at least about
0.01 mg/mL to about 0.25 mg/mL of the polyamide polymer or at least
about 0.1 mg/mL to about 1.0 mg/mL octenidine.
In some embodiments, the immunogenic composition (or other
preserved formulation) comprises between about 0.05 mg/mL and about
5 mg/mL, or between about 0.1 mg/mL and about 0.25 mg/mL of the
polyamide polymer or the octenidine.
In some embodiments, the immunogenic composition (or other
preserved formulation) comprises between about 0.1 mg/mL and about
0.25 mg/mL of the polyamide polymer or the octenidine.
In other embodiments, the immunogenic composition (or other
preserved formulation) comprises not less than about 0.1 mg/mL of
the polyamide polymer.
In still other embodiments, the immunogenic composition (or other
preserved formulation) comprises not less than about 0.1 mg/mL of
the octenidine.
In some embodiments, buffered solutions containing relatively lower
amounts of salts, particularly including phosphate salts, are added
to the immunogenic composition, or other preserved multi-dose
formulation, to improve the solubility of the octenidine. This is
based on Applicants' unexpected discovery that octenidine is less
soluble in routinely used phosphate buffered saline (PBS). To
address the problem of poor solubility, Applicants' tried a variety
of modifications and, ultimately determined that replacing the
sodium phosphate with sodium bicarbonate provided the required
solubility. As such, in some embodiments, multi-dose formulations
including octenidine contain less phosphate than standard PBS.
Accordingly, in some embodiments, the buffer may contain for 100 L
about 5 g of NaH.sub.2PO.sub.4 and about 200 g of Sodium
bicarbonate (instead of standard PBS, which contains about a 20:1
ratio of Na.sub.2HPO.sub.4 to KH.sub.2PO.sub.4).
In some embodiments, the buffer may contain relatively less NaCl,
with osmolality maintained by the compensatory addition of an
appropriate amount of glucose. The buffer may be further
supplemented with additional divalent ions, CaCL.sub.2 and
MgC12.
In some embodiments, the quantity of phosphate is generally reduced
50-fold, as compared to standard PBS, with bicarbonate substituting
for the phosphate.
In some embodiments, the immunogenic composition comprises an
adjuvant.
In an embodiment of the immunogenic composition, the antigenicity
of the immunogenic composition is stable for not less than 1 year,
1.5 years, 2 years or 2.5 years.
In general, a "stable" multi-dose formulation exhibits no
unacceptable levels of microbial growth, and substantially no or no
breakdown or degradation of the active biological or small molecule
component. As used herein, a "stable immunogenic composition" is a
preserved formulation that remains capable of eliciting a desired
immunologic response when administered to a target animal. Stable
immunogenic compositions often include those that lose no more than
one half of a log of activity during some specified time interval.
For example, "stable for at least one year" means that an
immunogenic composition with a beginning titer of about X Log 10
CCID50/dose of activity will retain at least about (X-0.5) Log 10
CCID50/dose of activity for at least one year after the preserved
immunogenic composition has been prepared (e.g. placed into its
sterile container; or, resuspended from a freeze-dried pastille
contained within a sterile vial).
In particular embodiments, the preserved multi-use formulations,
including preserved immunogenic compositions, remain stable for the
specified time with numerous repeated inoculations/insertions into
the multi-dose containers. In such cases, a term such as "stable
for at least one year with up to ten usages" may be used to
described a preserved formulation, which is contained within a
multi-dose container. In some preferred embodiments, the multi-dose
formulations remain stable even with 10, 15, 20, 30, 40, 50 or even
100 inoculations/insertions.
In an embodiment, following inoculation of the immunogenic
composition with one or more micro-organisms, the concentration of
said micro-organisms is reduced over time. As used herein,
"inoculation of the composition" refers generally to the
introduction into the composition of a foreign object (e.g. an
hypodermic needle), which may contain potentially contaminating
microorganisms. An example of inoculation is the repeated
introduction of needles into a vial containing a multi-dose vaccine
formulation. Another example is the repeated introduction of
needles into a vial containing a multi-dose vaccine formulation.
Each of the foregoing are examples of "inoculation," as used
herein.
In some embodiments, following inoculation of the immunogenic
composition with one or more bacteria strains, the composition
presents at least 1.0 log reduction from the initial micro-organism
count at 24 hours, at least 3.0 log reduction at 7 days from the
previous value measured and not more than 0.5 log increase at 28
days from the previous value measured.
In other embodiments, following inoculation of the immunogenic
composition, the composition presents at least 2.0 log reduction
from the initial calculated count at 6 hours after inoculation, at
least 3.0 log reduction at 24 hours from the previous value
measured and no recovery at 28 days. In some embodiments, the one
or more micro-organisms are selected from the group consisting of
P. aeruginosa, S. aureus, E. coli and B. subtilis.
In some embodiments, the composition is inoculated multiple times,
including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more times. Any number of inoculations above 1 are
envisioned by Applicants.
In some embodiments, a second inoculation occurs at 6 hours
following an initial inoculation, a third inoculation occurs at 24
hours following the initial inoculation, a fourth inoculation
occurs at 7 days following the initial inoculation and a fifth
inoculation occurs at 14 days following the initial
inoculation.
In other embodiments, the immunogenic composition comprises one or
more of a buffer, a cryoprotectant, a salt, a divalent cation, a
non-ionic detergent, and an inhibitor of free radical
oxidation.
In an embodiment, the disclosure provides a vial containing the
multi-dose immunogenic or other active-containing composition.
In an embodiment, the vial contains more than 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 doses of the
immunogenic composition.
In an embodiment, the disclosure provides a pre-filled vaccine
delivery device comprising a multi-dose formulation comprising
biologic or small molecule active ingredients. In some embodiments,
the device comprises a syringe. In other embodiments, the device
comprises a dual or multiple chamber syringe or vials or
combinations thereof. In yet other embodiments, the pre-filled
vaccine delivery device comprises a multivalent immunogenic
composition that is formulated for intramuscular or subcutaneous
injection.
In an embodiment, the container comprises two doses or more of the
multivalent immunogenic composition of claim 7, wherein each dose
comprises 0.1 to 2 mL of the composition. In some embodiments, the
composition comprises polyamide polymer at a concentration of
between about 0.1 mg/dose to about 0.25 mg/dose or between about
0.05 mg/dose and about 0.125, wherein the dose is a 0.5 mL
dose.
The following examples are simply intended to further illustrate
and explain the present invention. The examples, therefore, should
not be regarded as limiting the scope of the invention or manner in
which it may be practiced.
EXAMPLES
As described below, both the antimicrobial polyamide polymers and
octenidine were tested and demonstrated to be effective vaccine
privative agents. Table 2 presents a comparison of USP and EP
requirements for antimicrobial effectiveness testing for vaccine
preservative agents.
TABLE-US-00002 TABLE 2 USP and EP requirement for antimicrobial
effectiveness testing Time USP EP A EP B Requirements for bacterial
log reduction 6 h Not required 2 Not required 24 h Not required 3 1
7 d 1 No recovery 3 14 d 3 No recovery No increase 28 d No increase
No recovery No increase Requirements for fungal log reduction 7 d
No increase 2 No increase 14 d No increase No increase 1 28 d No
increase No increase No increase Meyer B. K. "Antimicrobial
Preservative Use in Parenteral Products: Past and Present," Merck
Research Laboratories. J. of Pharmaceutical Sciences, 2007
Tables 3 and 4 present the acceptance criteria/European
Pharmacopoeia (.sctn. 5.1.3) and European Pharmacopoeia for vaccine
for veterinary use.
TABLE-US-00003 TABLE 3 Acceptance criteria/European Pharmacopoeia
(Sec. 5.1.3) 6 h 24 h 7 days 14 days 28 days Bacteria 2 log 3 log
-- -- NR -- 1 log 3 log -- NI Fungi -- -- 2 log -- NI -- -- -- 1
log NI NR: No recovery NI: No increase in number of viable
micro-organisms compared to the previous reading
TABLE-US-00004 TABLE 4 Acceptance criteria/ European Pharmacopoeia
for vaccines for veterinary use 24 h 7 days 14 days 28 days
Bacteria NI 3 log NI Fungi -- NI NI NI: No increase in number of
viable micro-organisms compared to the previous reading
Example 1
Polyamide Preservative Study
Meyer et al. (J. Pharm. Sc. 96(12):3155-67; 2007) indicated the
following Minimum Inhibitory Concentration (MIC) data for
thimerosal: 4-8 .mu.g/mL (E. coli, Pseudomonas aeruginosa); 0.2
.mu.g/mL (Staphylococcus aureus); 32 .mu.g/mL (Candida albicans);
128 .mu.g/mL (Aspergillus niger). Further, the concentration of
thimerosal in various Merial veterinary vaccines is about 0.005% to
about 0.01% (=about 50 to about 100 .mu.g/ml). Based on the
foregoing, three different doses of the polyamide polymer were
selected as a starting point (0.1, 1 and 10 mg/mL). Applicants
could not predict in advance whether these concentrations would be
effective.
##STR00036## MW=7.6 kDa (see Table 1)
TABLE-US-00005 TABLE 5 Preservative efficacy of polyamide polymer
(A and B are criteria from the European pharmacopeia) P. S. aureus
aeruginosa C. albicans A. brasiliensis Dose (mg/mL) Criteria 0.1 1
10 0.1 1 10 0.1 1 10 0.1 1 10 A criteria + + + - B criteria + + + -
USP + + + + Veterinary + + + + use
Satisfactory Vet and USP were obtained with the three doses. No
low-dose effect observed, indicating the optimal dose is likely
less than 100 .mu.g/mL.
Methodology.
For each concentration of polyamide polymer, dilution
neutralization was carried out by inoculating 10 mL of test product
with 100 .mu.L of each test strain. Countings were performed at Do,
6 h (bacteria only), 24 h (bacteria only), D7, D14 and D28. For
each counting, 1 mL of the sample was neutralized in 9 mL of
neutralizing solution (10 minutes), for S. aureus, a second 1:10
dilution in the same solution. Then 1 mL of the mixture was
transferred in Petri dishes and covered with 15 mL of agar medium
(Trypcase Soy Agar for bacteria and Sabouraud Dextrosed Agar for
yeasts and molds). The countings were achieved after 24-48 hours
incubation at 32.5.degree. C..+-.2.5.degree. C. for bacteria, and
48-72 hours at 22.5.degree. C..+-.25.degree. C. for yeasts and
molds. The test micro-organisms included Staphylococcus aureus,
Pseudomonas aeruginosa, Candida albicans and Aspergillus
brasiliensis. Finally, the neutralization solution contained: Tween
80 (10%), lecithin (2%), saponin (2%), sodium thiosulfate (0.5%)
and buffered solution chloride. The saponin was from the bark of
Quillaja saponaria.
TABLE-US-00006 TABLE 6 Preservative efficacy of polyamide polymer
at 0.1 mg/mL A0202-32B - 0.1 mg/mL S. aureus P. aeruginosa C.
albicans A. brasiliensis Inoculum 8 .times. 10.sup.6 7.9 .times.
10.sup.5 1.7 .times. 10.sup.5 1.9 .times. 10.sup.5 T0 2 .times.
10.sup.3 4.9 .times. 10.sup.3 2.6 .times. 10.sup.2 4.1.0 .times.
10.sup.5 6 h <10 >5.3 log <10 >4.9 log - - 24 h <10
>5.3 log <10 >4.9 log - - 7 d <10 >5.3 log <10
>4.9 log <10 >4.2 log 9.7 .times. 10.sup.4 0.3 log 14 d
<10 >5.3 log <10 >4.9 log <10 >4.2 log 1.0
.times. 10.sup.5 0.3 log 28 d <10 >5.3 log <10 >4.9 log
<10 >4.2 log 6.6 .times. 10.sup.4 0.5 log A Criteria + + + -
B Criteria + + + - Veterinary Use + + + +
TABLE-US-00007 TABLE 7 Preservative efficacy of polyamide polymer
at 1.0 mg/mL A0202-32B - 1.0 mg/mL S. aureus P. aeruginosa C.
albicans A. brasiliensis Inoculum 8 .times. 10.sup.6 7.9 .times.
10.sup.5 1.7 .times. 10.sup.5 1.9 .times. 10.sup.5 T0 8 .times.
10.sup.3 4.9 .times. 10.sup.3 10 3.3 .times. 10.sup.5 6 h <10
>5.3 log <10 >4.9 log - - 24 h <10 >5.3 log <10
>4.9 log - - 7 d <10 >5.3 log <10 >4.9 log <10
>4.2 log 9.1 .times. 10.sup.4 0.3 log 14 d <10 >5.3 log
<10 >4.9 log <10 >4.2 log 9.6 .times. 10.sup.4 0.3 log
28 d <10 >5.3 log <10 >4.9 log <10 >4.2 log 6.3
.times. 10.sup.4 0.5 log A Criteria + + + - B Criteria + + + -
Veterinary Use + + + +
TABLE-US-00008 TABLE 8 Preservative efficacy of polyamide polymer
at 10 mg/mL A0202-32B - 10 mg/mL S. aureus P. aeruginosa C.
albicans A. brasiliensis Inoculum 8 .times. 10.sup.6 7.9 .times.
10.sup.5 1.7 .times. 10.sup.5 1.9 .times. 10.sup.5 T0 2.3 .times.
10.sup.5 3 .times. 10.sup.3 7.2 .times. 10.sup.2 3.9 .times.
10.sup.5 6 h <10 >4.3 log <10 >4.9 log - - 24 h <10
>4.3 log <10 >4.9 log - - 7 d <10 >4.3 log <10
>4.9 log <10 >4.2 log 4.4 .times. 10.sup.4 0.6 log 14 d
<10 >4.3 log <10 >4.9 log <10 >4.2 log 4.4
.times. 10.sup.4 0.6 log 28 d <10 >4.3 log <10 >4.9 log
<10 >4.2 log 1.8 .times. 10.sup.4 1 log A Criteria + + + - B
Criteria + + + - Veterinary Use + + + +
Example 2
Octenidine Preservative Study
Methodology.
For each concentration of octenidine, dilution neutralization was
carried out as disclosed in Example 1.
TABLE-US-00009 TABLE 9 Preservative efficacy of octenidine
dihydrochloride (1 mg/mL) Octenidine Dihydrochloride - 1 mg/mL S.
aureus P. aeruginosa C. albicans A. brasiliensis Inoculum 1.0
.times. 10.sup.6 1.2 .times. 10.sup.6 1.9 .times. 10.sup.5 1.9
.times. 10.sup.5 T0 <10.sup.2 <10 <10 1.6 .times. 10.sup.5
6 h <10.sup.2 >4 log <10 >5.1 log - - 24 h <10.sup.2
>4 log <10 >5.1 log - - 7 d <10.sup.2 >4 log <10
>5.1 log <10 >4.3 log 2.9 .times. 10.sup.4 0.8 log 14 d
<10.sup.2 >4 log <10 >5.1 log <10 >4.3 log 1.7
.times. 10.sup.4 1 log 28 d <10.sup.2 >4 log <10 >5.1
log <10 >4.3 log 1.4 .times. 10.sup.4 1.1 log A Criteria + +
+ - B Criteria + + + + Veterinary Use + + + +
TABLE-US-00010 TABLE 10 Preservative efficacy of octenidine
dihydrochloride (0.1 mg/mL) Octenidine Dihydrochloride - 0.1 mg/mL
S. aureus P. aeruginosa C. albicans A. brasiliensis Inoculum 1.0
.times. 10.sup.6 1.2 .times. 10.sup.6 1.9 .times. 10.sup.5 1.9
.times. 10.sup.5 T0 <10 <10 <10 1.7 .times. 10.sup.5 6 h
<10.sup.2 >5 log <10 >5.1 log - - 24 h <10.sup.2
>5 log <10 >5.1 log - - 7 d <10.sup.2 >5 log 50 4.4
log <10 >4.3 log 7.5 .times. 10.sup.4 0.4 log 14 d
<10.sup.2 >5 log <10 >5.1 log <10 >4.3 log 1.0
.times. 10.sup.4 1.3 log 28 d <10.sup.2 >5 log <10 >5.1
log <10 >4.3 log 2.8 .times. 10.sup.4 0.8 log A Criteria + +
+ - B Criteria + + + + Veterinary Use + + + +
TABLE-US-00011 TABLE 11 Preservative efficacy of octenidine
dihydrochloride (0.01 mg/mL) Octenidine Dihydrochloride - 0.01
mg/mL S. aureus P. aeruginosa C. albicans A. brasiliensis Inoculum
1.0 .times. 10.sup.6 1.2 .times. 10.sup.6 1.9 .times. 10.sup.5 1.9
.times. 10.sup.5 T0 10 <10 2 .times. 10.sup.3 2.8 .times.
10.sup.5 6 h <10 >5 log <10 >5.1 log - - 24 h <10
>5 log <10 >5.1 log - - 7 d <10 >5 log <10 4.4
log <10 >4.3 log 3.8 .times. 10.sup.4 0.7 log 14 d <10
>5 log <10 4.4 log <10 >4.3 log 3.2 .times. 10.sup.4
0.8 log 28 d <10 >5 log <10 4.4 log <10 >4.3 log
3.1.0 .times. 10.sup.4 0.8 log A Criteria + + + - B Criteria + + +
- Veterinary Use + + + +
TABLE-US-00012 TABLE 12 Preservative efficacy of octenidine
dihydrochloride (0.001 mg/mL) Octenidine Dihydrochloride - 0.001
mg/mL S. aureus P. aeruginosa C. albicans A. brasiliensis Inoculum
10 .times. 10.sup.6 1.2 .times. 10.sup.6 1.9 .times. 10.sup.5 1.9
.times. 10.sup.5 T0 1.6 .times. 10.sup.5 7.5 .times. 10.sup.4 1.7
.times. 10.sup.5 2.4 .times. 10.sup.5 6 h 1.6 .times. 10.sup.2 3.8
log 1.0 .times. 10.sup.3 3 log - - 24 h <10 >5 log 1.0
.times. 10.sup.4 3 log - - 7 d 2 .times. 10.sup.2 3.7 log >3.3
.times. 10.sup.6 0 log 20 4 log 1.7 .times. 10.sup.5 0 log 14 d 1.4
.times. 10.sup.2 3.9 log >3.3 .times. 10.sup.6 0 log 10 4.3 log
1.4 .times. 10.sup.5 0.1 log 28 d 40 4.4 log >3.3 .times.
10.sup.6 0 log <10 >4.3 log 1.0 .times. 10.sup.5 0.3 log A
Criteria - - + - B Criteria + - + - Veterinary Use + - + +
TABLE-US-00013 TABLE 13 Summary of the preservative efficacy of
octenidine S. aureus P. aeruginosa C. albicans A. brasiliensis Dose
(mg/mL) Criteria A B C D A B C D A B C D A B C D A criteria + + + +
- - - - B criteria - - - + + USP - + Veterinary use - A = 0.001
mg/mL B = 0.01 mg/mL C = 0.1 mg/mL D = 1 mg/mL
TABLE-US-00014 TABLE 14 Overview of polymer and octenidine as
vaccine preservatives Criteria Pol(imido-amine) Octenidine Efficacy
MIC .ltoreq.0.25 .mu.g/ml MIC .ltoreq.1 .mu.g/ml Broad spectrum
Broad spectrum Non antibiotics active Non antibiotics active
Innocuity Cytotoxicity data and Cytotoxicity data failed to pretox
IV/IP/oral on mouse/rat demonstrate any adverse MTD around 10 to 25
mg/kg effect to wound healing IC.sub.50 hemolysis >5000 mg/ml
LD.sub.50 on rat = 10 mg/kg Favorable therapeutic index Food safety
Absence of MRL MRL assessment Oral, IP and IV toxicity data
(cutaneous treatment) available ADI = 37.5 .mu.g/person
EMA/CVMP/735219/2009 MTD was evaluated by IP, IV, and oral in mouse
and rat; and by intramammary in cow (see US 2014/0271526 (to
Merial, Inc.) and US 2014/0275469 (to Genzyme)). Therapeutic Index
(TI) was estimated as a multiple of the MIC for the Mouse (IP)
MTD.
Example 3
Oil-in-Water Emulsion Preservative Study
Methodology.
For each concentration of preservative (or preservative
combination), dilution neutralization and other testing was carried
out as disclosed in Example 1. The "TS6" formulation is an
oil-in-water emulsion, described in U.S. Pat. No. 7,371,395 B2 (to
Merial, Inc.; see especially Example 1). For each formulation (see
Table 15), the TS6 antigen component consisted of porcine
circovirus 2 (PCV2) virus-like particles (VLPs) and inactivated
Mycoplasma hyopneumoniae (M. hyo) (see U.S. Pat. No. 7,371,395 B2,
Examples 3 and 5).
TABLE-US-00015 TABLE 15 Summary of test conditions and results
Human Veterinary Pharmacopeia Criteria Pharmacopeia ID Formula
Preservative S. aureus P. aeruginosa C. albicans A. brasiliensis
Crit. A Crit. B A TS6 POL 250 .mu.g/ml + + + + + + B TS6 POL 500
.mu.g/ml + + + + + + C TS6 POL 100 .mu.g/ml + + + + + + +
Thiomersal 25 .mu.g/ml D TS6 Thiomersal 25 .mu.g/ml + + + + -* -* E
TS6 OCT 250 .mu.g/ml + - + + -* - F TS6 POL + OCT 100/100 .mu.g/ml
+ + + + - + *for S. aureus
Taken together, the data presented in Examples 1 to 3 (and
throughout this disclosure) indicate that both the polyamide
polymer and octenidine are safe and effective vaccine
preservatives. Finally, Example 3 demonstrates that the polyamide
polymers and octenidine are effective preservatives in a
significantly challenging vaccine environment: oil-in-water
emulsion. For example, this complicated formulation system presents
many opportunities for unwanted interactions with proteins and
surfactants, which could have impaired the preservative efficacy of
the polyamide polymers and/or the octenidine. In fact, in a
previous experiment Applicants showed that 100 .mu.g/ml of either
the polyamide polymer or the octenidine alone did not exhibit
satisfactory preservative efficacy in this oily vaccine
environment. Accordingly, it was quite unexpected to observe the
positive results summarized in Table 15. And finally, the
antimicrobial polyamide polymers demonstrated unexpected superior
efficacy over the octenidine and the mercurial preservative,
Thiomersal, in the oily vaccine environment.
Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above examples is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
* * * * *